ABSTRACT In response to NINDS PA-18-358 (?exploratory and innovative projects? related to neurological disorders), we propose a 2-yr project to develop a novel method to electrically stimulate the spinal cord. The purpose is to restore respiratory muscle activation and breathing during acute or chronic hypoventilation associated with opioid overdose or neurotrauma. Based on deep brain stimulation data, the method is called temporal interference (T-I) stimulation. The premise is to target the spinal cord with two, high frequency, but low amplitude electrical waveforms. The waveforms are delivered at kilohertz frequencies that are well above values that directly stimulate neurons. The frequency of the two waveforms is offset by a small amount (e.g. 1- 5 Hz), and where the two electrical fields sum in the tissue, neuronal populations are recruited in phase with the offset. Our overall hypothesis is that T-I stimulation can be used to target energy to the ventral cervical spinal cord to regulate diaphragm activation with minimal off target effects. For this application, we conducted proof-of-concept preliminary experiments using rat models of opioid overdose and cervical spinal cord injury (SCI). We initially examined T-I stimulation delivered via simple sub-cutaneous neck electrodes following acute opioid overdose. The rationale is that a rapid and easily applied method to sustain breathing could be useful to sustain ventilation in emergency clinical situations. Remarkably, T-I stimulation delivered using sub- cutaneous leads in the neck region was able to evoke diaphragm motor recruitment, and the discharge could be regulated by varying the offset frequency between the two stimulus waveforms. To our knowledge, no prior methods have been able to induce rhythmic diaphragm contractions i.e., ?pacing? with a minimally invasive stimulation approach. Additional preliminary experiments focused on epidural stimulation of the cervical spinal cord. The rationale is that epidural stimulation is gaining traction as a means of restoring somatic and/or autonomic motor function after SCI and shows promise for activating the diaphragm. Electrodes on the mid- cervical dorsal epidural surface were used to deliver two waveforms at KHz frequencies. The T-I dual wave epidural stimulation was remarkably effective at activating and regulating diaphragm motor units. Compared to single wave epidural stimulation, we predict that T-I will offer advantages including 1) improved efficacy after incomplete and/or chronic lesions; 2) ability to produce a wide range of diaphragm motor unit recruitment patterns with more natural ?burst? envelope, and 3) ability to more focally target the stimulus to the ventral horn. The overall hypothesis will be tested by evaluating and optimizing T-I stimulation delivered via subcutaneous (Aim 1) or epidural electrodes (Aim 2). The project is innovative since dual waveform T-I stimulation has not previously been explored as a means of activating the respiratory muscles. PI Dr. Fuller has extensive experience with rodent models of SCI and Co-I Dr. Otto is a biomedical engineer with 20+ years of experience in electrical stimulation of the central nervous system including electrode design.